The Josephin domain-containing proteins are one of the five major families of deubiquitinating enzymes (DUBs). The founding member of the family is ataxin-3, a polyglutamine protein and the causative agent of the most common inherited ataxia, spinal cerebellar ataxia type 3 (SCA3, also known as Machado-Joseph disease). Ataxin-3's DUB activity resides in the molecule's N-terminal half, the so-called Josephin domain. Ataxin-3 is linked to many cellular processes that are related to maintaining protein homeostasis, including the transport of misfolded proteins to aggresomes and the flux of proteins through the endoplasmic reticulum- associated degradation pathway (ERAD). Ataxin-3 can also suppress the toxicity of expanded repeat polyglutamine domains. All of these functions require DUB activity, establishing the biological significance of the Josephin domain and hinting at a link between Josephin DUB activity and neurodegeneration induced by protein misfolding. Three homologs of ataxin-3 are encoded in the human genome: The ataxin-3-like protein (AT3L), Josephin-1, and Josephin-2. All three possess DUB activity, but differ substantially in catalytic efficiency and specificity. Josephin domain-containing homologs are conserved in most eukaryotes, including plants and protozoans, implying that this domain and the DUB activity it expresses are responsible for valuable biological functions. We propose to analyze the molecular structure and function of the human Josephin domain-containing proteins in order to understand how they recognize and act upon substrates and other interacting proteins. We will use X-ray crystallography to determine structures for different Josephin domains, alone and in complex with ubiquitin substrates; characterize the DUB activities of the four human Josephin domain-containing proteins, assessing how they bind and cleave both small and large ubiquitin substrates; explore their ubiquitin- binding activities; and probe the interactions of ataxin-3 with its cellular binding partners.